This invention relates to a composition that inhibits the immunoglobulin E (IgE) antibody production and response in vivo. It also relates to the use of such composition in the prophylaxis and treatment of type I allergy-related diseases.
Allergies are classified into I to IV types, and the type I allergy is frequently observed and brings about bronchial asthma, some urticarialy, allergic rhinitis, anaphylaxis etc. Type I allergy is a biological reaction in which IgE antibody is involved, and the mechanism of this type of allergy consists of (1) production of IgE antibody by immunization of a living body with an antigen, (2) binding of the IgE antibody via IgE receptor to mast cells and basophils, (3) upon re-invasion of the antigen, it binds to the previous IgE antibody bound to the surfaces of mast cells and basophils, thereby forming cross-linkages between IgE receptors on these cells, (4) via various reactions such as inflow of Ca ion, chemical mediators such as histamine, leukotriene, eosinophil chemotactic factor of anaphylaxis (ECF-A), platelet activating factor (PAF) are released to develop the allergic symptom.
The majority of known anti-allergy agents are anti-histamine agents or inhibitors of the release of chemical mediators. It is expected that the type of drugs whose working mechanism is based on the inhibition of the earliest step of occurrence of type I allergy, i.e. production of IgE antibody, can be a superior drug against this type of allergies, but as such a type of drug, there is no drug with sufficient efficacy. IPD (IPD-1151T, Suplatast Tosylate) is the only anti-allergy agent having some ability to-inhibit the production of IgE antibody (Bio Industry, Vol. 13, No. 12, pp. 42-50 (1996)), and this compound has extremely weak inhibitory action on the production of IgE antibody.
It is, therefore, an object of the present invention to provide a composition which inhibits the IgE antibody production and response and which is more potent than known agents or compositions in the anti-IgE antibody production inhibitory activity,.
According to the present invention, the above and other objects may be accomplished by providing a composition for inhibiting the IgE antibody production and response comprising a capsule component of Klebsiella oxytoca or Klebsiella pneumoniae, a fragment thereof produced by the treatment of said capsule component with an acid, a base or a reducing agent.
Particularly preferable Klebsiella oxytoca is Klebsiella oxytoca strain TNM3 (FERM BP-4669, deposited with the National Institute of Bioscience Human-Technology, Agency of Industrial Science and Technology, the Ministry of International Trade and Industry, Japan) or mutants thereof. Particularly preferable Klebsiella pneumoniae is Klebsiella pneumoniae strain K19 (Michel Beurret et al., xe2x80x9cStructural investigation of the capsular polysaccharide from Klebsiella K19 by chemical and N.M.R. analysesxe2x80x9d, Carbohydrate Research, 157:13-25 (1986)) or mutants thereof.
These mutants can be generated by known mutagenesis means including exposure of Klebsiella oxytoca strain TNM3 or Klebsiella pneumoniae strain K19 to radiation such as ultraviolet light, X-ray etc. or contacting it with chemical mutagens such as ethyl methane sulfonate (EMS), N-methyl-Nxe2x80x2-nitro-N-nitrosoguanidine (MNNG) etc.
The presence or absence and the degree of inhibitory activity on IgE antibody production, of the capsule component of these microorganisms or a fragment thereof obtainable by treating the capsule component with acid, base or a reducing agent can be easily measured in the methods described below.
Klebsiella oxytoca strain TNM3 and Klebsiella pneumoniae strain K19 have polysaccharides as a capsule component having the following linkage mode and constituent molar ratio. That is, the capsule component is a polysaccharide having a repeating unit represented by the following formula: 
wherein L-Rha is an L-rhamnose residue, D-Gal is a D-galactose residue, D-Glc is a D-glucose residue, and D-GlcUA is a D-glucuronic acid reside, and the numerical values in the small brackets indicate the positions of the glycoside linkage, and the proportions of respective monosaccharide residues constituting the polysaccharide chain in terms of moles are 0.8 to 1.2 for 
The above polysaccharide of fragments thereof produced by the treatment of the polysaccharide with an acid, a base or a reducing agent exhibit a potent inhibitory activity against IgE antibody production and response in vivo. Therefore, they are useful in the prophylaxis and treatment of type I allergy-related diseases.
The capsular polysaccharide component is prepared by culturing the microorganism in a medium, wet sterilizing or ultrasonificating the microorganism to release the capsular component into the medium, centrifuging the culture to give a supernatant, ultrafiltering the supernatant through a membrane having a fractionation molecular weight of 1xc3x97103 such as available from Millipore. The polysaccharide occurs in the retainant fraction.
The product resulting from the fragmentation of the capsule component can be obtained by treating the capsule component with an acid, a base or a reducing agent. Further, the treatment with a reducing agent can also be carried out by adding ferrous sulfate and EDTA to the medium while the microorganism is cultured.
Klebsiella oxytoca strain TNM3 is particularly preferable in the present invention. Culture of microorganisms of the genus Klebsiella can be carried out generally according to the following method. That is, the medium for culture may be any medium insofar as the microorganisms of the genus Klebsiella can grow in it, and carbon and nitrogen sources, inorganic salts, and trace nutrient sources which are necessary for producing the desired capsule component are contained in it, and there is no other limitation to the medium.
The carbon source used may be e.g. glucose, lactose, maltose, xylose, mannitol, sucrose, rhamnose, arabinose, trehalose, raffinose etc. The nitrogen source used may be e.g. synthetic compounds such as nitrates, ammonium salts, urea etc., natural organic matter such as polypeptone, corn steep liquor, yeast extract, meat extract, delipidated soybean extract, peptide, amino acid etc. The inorganic salts used may be e.g. phosphates, potassium salts, sulfates, magnesium salts etc. The trace nutrient sources used may be e.g. yeast extract, various vitamins etc. As necessary, calcium salts, manganese salts etc. may be added to the medium.
The medium used may be a solid or liquid medium. If the liquid medium is used, stationary culture may be used, but shake culture or shake culture under aeration is more preferable to achieve the desired capsule component in higher yield. The pH of the medium during culture is not particularly limited inasmuch as the pH is suitable for growth of the microorganism and simultaneously does not prevent the target capsule component from being produced. However, the pH range of 4 to 8 is usually preferable. The culture temperature is not particularly limited, but usually 20 to 35xc2x0 C. is preferable. The period of culture is suitably determined so as to maximize the production of the target capsule component, but 1 to 7 days are usually preferable.
The resulting culture itself can be used as an inhibitor of the production of IgE antibody after sterilization without purification. However, it is more preferable to remove the microorganism and to purify the desired capsule component. The removal of the microorganism can be carried out by releasing capsules from the microorganism by wet-sterilization, and subsequent centrifugation and/or filtration in a usual manner. After removal of the microorganism, the culture may be further subjected to purification procedures: that is, a water miscible organic solvent such as methanol, ethanol, isopropanol, acetone or the like is added to the culture to form precipitates which are then dissolved in water, dialyzed against water and the dialysate is dried by such means as air drying, drying in hot air, spray drying, drum drying, drying under reduced pressure, lyophilization etc. For purification, other methods may be carried out. For example, use can be made of ultrafiltration (e.g. a membrane with a cut off molecular weight of 1xc3x97103, manufacture by Millipore can be used for concentration) followed by drying the resulting concentrate, and as necessary, various kinds of column chromatography, such as ion exchange, gel filtration, and affinity and precipitation or salting-out with quaternary ammonium salts, precipitation with organic solvent etc. are also used.
The culture itself or the purified capsule component may be further subjected if desired to various treatments including addition of a reducing agent, acid or base hydrolysis, heating under pressure, ultrasonication etc. For acid or base hydrolysis, an acid such as sulfuric acid, hydrochloric acid etc. or a base is added preferably under regulated conditions to the culture after culturing the microorganism or to the capsule component purified as described above.
For culture, a reducing agent may be added to the medium to such a level as not to decrease the amount of capsule component produced in the medium. As the reducing agent, a combination of ferrous sulfate and/or ferrous chloride and EDTA is preferable.